Biology Reference
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obvious that some of these regulatory connections play more critical roles in
development and physiology than many others do. It seems likely that the
same consideration applies to miRNAs.
It is common for regulatory proteins to be recycled during developmen-
tal and physiological processes, and it is similarly observed that individual
miRNAs function in multiple settings (
Smibert and Lai, 2010
). A curious
emerging theme is that many miRNAs exhibit different key targets in
different locations. For example, excess brain apoptosis and associated
behavioral defects in
mir-8
mutants are suppressed by heterozygosity for
the direct target
atrophin
, encoding a transcriptional coactivator (
Karres
et al
.,
2007
). However, miR-8 also functions in the fat body to activate insulin
signaling by repressing the zinc finger protein encoded by
u-shaped
(
Hyun
et al
., 2009
). Knockdown of
u-shaped
, but not
atrophin
, in the fat body
rescued
mir-8
mutant phenotypes. In another example, miR-14 directly
restricts the activity of the nuclear receptor encoded by the
Ecdysone receptor
during metamorphosis (
Varghese and Cohen, 2007
), whereas it also func-
tions in insulin neurosecretory cells of the adult brain to directly repress
sugarbabe
, a zinc finger protein that regulates insulin gene expression
(
Varghese
et al
., 2010
). It is plausible to consider that miRNAs may have
so many targets, in part due to their frequent acquisition of new compelling
functions in different settings via novel target genes.
4.4. Dominant modifier screens for miRNA interactors
In most cases, directed tests for genetic interactions in a homozygous mutant
background require the generation of complicated stocks or recombinant
animals, which limits the throughput of analysis. However, one can exploit
this test in forward genetic screening by starting with a dominant pheno-
type. A simple way to do this is by sensitizing the animal by misexpressing a
gene to generate a mutant phenotype and then to ask whether the removal
of one copy of any other gene can modify this phenotype (
Fig. 8.3
B). This
approach, termed dominant enhancer/suppressor screening, was codified in
studies of the Ras signaling pathway 20 years ago (
Simon
et al
., 1991
). Here,
misexpression of oncogenic Ras in the developing
Drosophila
eye yielded a
rough eye, presumably due to hyperactivation of its downstream pathway.
Loci for which removal of one gene copy reduced the activity of oncogenic
Ras identified several downstream members of the pathway (
Simon
et al
.,
1991
) and helped to elucidate the signaling mechanism of this important
cancer pathway. Since effects of removing only one gene copy are assessed,
dominant enhancer/suppressor screening can be executed on a massive
scale, as illustrated by the subsequent examination of 850,000 mutagenized
animals in the
Ras1
eye screen (
Karim
et al
., 1996
), which collectively hit
most of the key components of Ras signal transduction. Thus, dominant
modifier screening is genuinely a “genome-wide” technique. Such an
